Antibiotic bl580

ABSTRACT

This disclosure describes two new antibiotics, designated BL580 Alpha and BL580 Beta , produced in a microbiological fermentation under controlled conditions using a new strain of Streptomyces hygroscopicus and mutants thereof. The new antibiotics are active antimalarial and anticoccidial agents.

United States Patent 1191 Martin et al.

[ ANTIBIOTIC BL580 [75] inventors: John Henry Edward James Martin,

New York, N.Y.; Sidney Kantor, Lawrence Township, NJ.

[73] Assignee: American Cyanamid Company,

Stamford, Conn.

22 Filed: Nov. 24, 1972 21 Appl. No.: 309,383

52 11.5. C1 424/121, 424/122, 195/80 51 1111. c1 A611 21/00 [58] Fieldof Search 424/121, 122; 195/80 [56] References Cited UNITED STATESPATENTS 3,595,955 7/1971 B081 et ai. 424/121 1111 3,812,249 [451 May21,1974 3,699,223 10/1972 Bergy et a] 424/121 Primary Examiner-Jerome D.Goldberg Attorney, Agent, or Firm-Edward Conroy, Jr.

[ 5 7 ABSTRACT This disclosure describes two new antibiotics, designatedBL580a and BL580/3, produced in a microbiological fermentation undercontroliled conditions using a new strain of Streptomyces hygroscopicusand mutants thereof. The new antibiotics are active antimalarial andanticoccidial agents.

4 Claims, 2 Drawing Figures America, Chicago, Illinois. Descri 1AETIBIOTIC eras-am BRIEF SUMMARY OF THE INVENTION DETAILED DESCRIPTIONOF THE INVENTION The new antibiotics which we have designated BL580a,and BL58OB areformed during the cultivation under controlled conditionsof a new strainof Streptomyces hygroscopicus. This new antibioticproducing strain was isolated from a soil sample collected in Iowa. Aviable culture of the new microorganism has been deposited with theCulture Collection Laboratory, Northern Utilization Research andDevelopment Division, United States Department of Agriculture, Peoria,Illinois, and has been added toits permanent collection. It is freelyavailable to the-public in this depository under its accession numberNRRL 5647.

The description and identification of this new microorganism, maintainedin the culture collection of the Lederle Laboratories Division, AmericanCyanamid Company, Pearl River, NY. as Culture No. BL580, was supplied byDr. H.'D. Tresner of these laboratories. The following is a generaldescription of the microorganism Streptomyces hygroscopicus NRRL 5647,based on diagnostic characteristics observed. Observations were made ofthe cultural, physiological, and morphological features of themicroorganism in accordance with the methods detailed by Shirling andGottlieb, In-

ternat. Journ. of Syst. Bacetiol. 16:313-340 (1966).

The underscored descriptive colors and color chip designations are takenfrom Jacobson et al., Color Harmony Manual, 3rd edit (1948), ContainerCorp. of

ptive details are recorded in Tables I through IV below.

Amount of Growth langiform, 0.6-0.7

paste-oatmeal and potato-dextrose agars; moderate onasparagine-dextrose, Hickey and Tresners inorganic salts-starchandBennetts agars; light on Czapeks solution agar.

Aerial Mycelium 7 Aerial Mycelium whitish to yellowish, becoming grayishin sporulation zones ranging from Fawn (4 ig) to Beaver (4 li) to Ashes(5 fe). Sporulation zones becoming black and hygroscopic in oldercultures. Soluble Pigments None on most media; yellowish on yeastextract, Bennetts and potato-dextrose agars and only in light amounts.Reverse Color Generally in yellowish shades on most media. MiscellaneousPhysiological Reactions Nitrates reduced to nitrites; completeliquefaction of gelatin; no formation of melanoid pigments onpeptone-iron agar; completepetonization of purple milk in 7 d s; tot;ansesit a insr wrhmd n g 2p cent but l0 percent. Carbon sourceutilization according to the method of Pridham and Gottlieb, J.Bacteriol. 56:107-1 14(1948) asfollows: Good utilization of adonitol,g-galactose, d-fructose', d-raffinose, salicin, d-xylose and dextrose;poor or no utilization of dmelezitose, d-melibiose, 1 -ar alaino ei-inositol, lactose, d-mannitol, I-dLE'lIIEEQS C sucrose andd-trehalose. Micromorphology Aerial mycelium gives rise to spore-bearingbranches which terminate in tightly coiled spirals of several turns;spores are mostlyisodiametric, cylindrical, phapm X 0.7 0.8 ,um. Sporessmooth as determined by electron microscopy; spore sheaths finelywrinkled.

I On the basis of the general characteristics observed,

microorganism BL580 is a member of a'large group of steptomycetescharacterized by gray spores, spiral spore chains, smooth-walled sporesand lack of melanin pigments. The hygroscopic nature of the culturealong with its entire composite of morphological and physiologicalcharacteristics makes it a representative strain of Streptomyceshygroscopicus as defined by H. D. Tresner and E. J. Backus, A BroadenedConcept of the Characteristics of Streptomyces hygroscopicus, Appl.Microbiol, 4:243-250 (I956) and H. D. Tresner,,E. J. Backus and J. A.Hayes, Morphological Spore Types in the Streptomyces hygroscopicusdike-Complex, Appl. Microbiol. 15:637 639 (1967).

TABLE I Cultural Characteristics of Strep Incubation: 14 daysTemperature: 28C.

Amount Soluble of Medium Growth Aerial Mycelium and/or Spores PigmentReverse Color Remarks Czapek's solution Light Trace of whitish aerialNone Colorless agar mycelium; no sporulation. to whitish Yeast extractGood Aerial mycelium whitish, Yellowish, Bamboo (2 tb) Blackishhygroscopic agar becoming Fawn (4 ig) to Beaver light areas in centralcolony (4 li) in sporulation zones. zones. Sporulation heavy. Kuster'soat Good Aerial mycelium whitish, None Lt. mustard Blackish hygroscopicflake agar becoming Fawn (4 ig) to Beaver tan (2 ie) areas in centralcolony (4 li) in sporulation zones. zones. Yellowish Sporulation heavy.exudate in marginal zones. Asparagine- Moderate Aerial mycelium whitish,None Bamboo (2 lb) Blackish hygroscopic dextrose agar (4 ig) insporulation areas.

PQWIQt mar na e.

becoming Ashes (5 fe) to Fawn areas in central colony zones.

TABLE EQQQEEWQ Cultural Characteristics of Srrepmmyces hygrmcopicus NRRL5647 Incubation: l4 days Temperature: 28C.

Amount Soluble of Medium Growth Aerial Mycelium and/or Spores PigmentReverse Color Remarks Hickey and Moderate Aerial mycelium whitish toNone Bamboo (2 fb) Extensive hygroscopic Tresners agar yellowish,becoming Fawn areas in central colony (4 ig) to Beaver (4 li) in zones.sporulation zones.

Sporulation heavy.

Inorganic salts- Moderate Aerial mycelium whitish to None Pastel yellowBlackish hygroscopic starch agar yellowish, becoming Fawn (1 db) areasin central colony (4 ig) to Beaver (4 ii) in zones. sporulation zones.

,Sporulation heavy.

Tomato paste- Good Aerial mycelium whitish to None Yellow MapleExtensive hygroscopic oatmeal agar yellowish, becoming Fawn (3 ng) areasin central colony (4 ig) to Beaver (4 li) in zones. Yellowishsporulation zones. exudate in marginal zones. Sporulation very heavy.

Bennett's agar Moderate. Aerial mycelium whitish, Yellowish, Bamboo (2fb) Blackish hygroscopic becoming Beaver (4 li) in light areas incentral colony sporulation zones. zones.

. Sporulation heavy Potato-dextrose Good Aerial mycelium whitish toYellowish, Yellow Maple Blackish hygroscopic agar. yellowish, becomingAshes light (3 ng) areas in central colony (5 fe) to Fawn (4 ig) inzones. sporulation zones.

Sporulation moderate.

TABLE II Micromorphology of Sl'reptomyces hygroscopic-us NNRL 5647Aerial Mycelium and/or Medium Sporiferous Structures Spore Shape SporeSize Spore Surface Kusters Aerial Mycelium gives Spores are mostly0.6-0.7 am Smooth as deter- Oatflake agar rise to spore bearingisodiametric, Y mined by electron branches which termincyclindrical, Xmicroscopy. Spore ate in tightly coiled phalangiform. sheaths finelyspirals of several 0.7-0.8 um wrinkled. turns.

TABLE III Miscellaneous Physiological Reaction of Streptomyceshygroscupicus NRRL 5647 Temperature: 28C.

Medium Incubation Period Amount of Growth Physiological Reaction OrganicNitrate Broth Organic Nitrate Broth Gelatin Peptone-iron Agar Purplemilk Yeast extract agar plus (4, 7,10 and 13%) NaCl 7 days l4 days 7days 24-48 hours 7 days it) days Good Good Good Good Good Good Nitratesreduced to nitrites Nitrates reduced to nitrites Complete liquefactionNo melanoid pigments produced Complete peptonization NaCl tolerance 7%but TABLE IV Carbon Source Utilization Pattern of S Ireptom yces it ygmsmpicus NR R L 5 647 Carbon Source Utilization Adonitol I-ArabinoseDextran dFructose i-lnositol Lactose d-Mannitol d-Melezitose d-Melihiosed'Raffinose l-Rhamnose Salicin Sucrose d-Trehalose 490 0 DextroseNegative Control 3 Good Utilization 2- Fnir Utilization l- PoorUtilization 0- Nu Utilization THE FERMENTATION PROCESS Cultivation ofthe microorganism Strepmmyces hygroscopicus NRRL 5647 may be carried outin a wide variety of liquid culture media. Media which are useful forthe production of the novel antibiotics BL580 1 and BL58OB include anassimilable source of carbon such as starch, sugar, molasses, glycerol,etc.; an assimilable source of nitrogen such as protein, proteinhydrolysate, polypeptides, amino acids, corn steep liquor, etc; andigorganic anions and cations, such as potassium, so-

5 dium, calcium, sulfate, phosphate, chloride, etc. Trace elements suchas boron, molybdenum, copper, etc. are supplied as impurities of otherconstituents of the media. Aeration in tanks and bottles is provided byforcing sterile air through or onto the surface of the fermentingmedium. Further agitation in tanks is provided by a mechanical impeller.An antifoaming agent such as l percent octadecanol in lard oil may beadded as needed.

INOCULUM PREPARATION Shaker flask inoculum of Streptomyces hygroscopicusNRRL 5647 is prepared by inoculating 100 ml. of sterile liquid medium in500 ml. flasks with scrapings or washings of spores from an agar slantof the culture. The following medium is ordinarily used:

Glucose 20 gm Soy flour l gm Corn steep liquor gm. Calcium carbonate3gm. Water to l()()0 ml The flasks are incubated at a temperature from25 to 29C., preferably 28C., and agitated vigorously on a rotary shakerfor 30 to 48 hours. These 100 ml. portions of inocula are used toinoculate one liter and 12 liter batches of the same medium in 2 literand liter glass fermentors. The inoculum mash is aerated with sterileair while growth is continued for 30 to 48 hours. These batches ofinocula are used to inoculate tank fermentors.

TANK FERMENTATION For the production of antibiotics BL580a and BL580B intankfermentors the following fermentation medium is preferably used.

Cornstarch 40 gm. Soy flour 10 gm. Corn steep liquor 5 gm. Calciumcarbonate 3 gm. Water to 1000 ml.

Each tank is inoculated with 3 to 10 percent of inoculum madeasdescribed above. Aeration is supplied at the rate of 0.5 to 1.0 liter ofsterile air per liter of broth per minute and the fermentation mixtureis agitated by an impeller driven at 200-400 rpm. The temperature ismaintained at to 29C., usually at 28C. The fermentation is ordinarilycontinued for 90 to 120 hours, at which time the mash is harvested.

PURIFICATION PROCEDURE solvent isallowed The crude oily residue from theethyl acetate extraction is dissolved in a minimal quantity of methylenechloride and allowed to seep into the column gel. The column is treatedwith methylene chloride followed by methylene chloride-ethyl acetate(1:1 v/v). A total of 49 volumes ml. per volume) is collected. Aseparate paper (one-fourth inch diameter) is dipped into each fraction,air dried and bioautographed on an agar plate seeded with Streptococcuspygge nes NYS. Fractions 7 to 37 comprising the active band, arecombined and concentrated to a residue.-

A two-phase system is prepared by mixing hex anezethyl acetate:methanolzwater (2812:1611). Acid washed diatomaceous earth is wet tedwith a portion of the aqueous phase and packed in increments into aglass column. The charge consists of a mixture of diatomaceous earth,lower phase and partially purified residuefrom the silica gelchromatographyThe column is developed with the upper phase of thesystem. Fractions of 20 ml. each are collected. The active fractions aredetermined as described above. Fractions 4 to 14 inclusive, containingBL580a are combined and concentrated to a residue. Similarly, fractions24 to 65,

comprising BLSSOB are concentrated to a residue. Amorphous, essentiallypure BLS a may be. obtained from the above BL580a residue from apartition column by solvent countercurrent distribution using a systemcomposed of hexane:ethyl acetatezmethanolzwater (56:12:3213). Amorphous,essentially pure BLSBOB may be obtained by subjecting the BL580/3 bandfrom a partition column to solvent countercurrent distribution usinghexane:ethyl acetate: methanol water (280:70:160:7).

Antibiotics BL580a and BL580B exhibit antimicrob a ctivity asruttst.teetqqq wseyrg esltlYfi at a concentration of 2.5 meg/ml. when assayed byconventional agar dilution techniques.

The usefulness of these new antibiotics is demonstrated by their abilityto control systemic lethal infections in mice. Both BL5800: and BLSSOBexhibited in vivo antimalarial activity in mice against Plasmodiumberghei according to the following test. Carworth Farms CF-l mice wereinoculated with l2 million parasitized (Plasmodium berghei) red cellsper mouse, randomized and caged in groups of5 to 10 mice. Drugdiettreatment was initiated within l-3 hours postinoculation and wascontinued for 6 successive days (day 05 postinoculation). Appropriategroups of untreated controls, both infected and uninfected, wereincluded and appropriate infected controls, treated with chloroquine orpyrimethamine, were also included. Determination of group (i.e. mean)mouse weights were made on the day of inoculation and on days 3 and 6postinoculation. The food was weighed before and immediately after thetreatment period and mean daily intakes of the compounds weredetermined. Geimsa-stained thin blood films were prepared once from 3-10mice in each treated group on day 7-l2 postinoculation and the meanparasitemia was compared with that of appropriate untreated controls.Mortality was recorded daily, with an observation period of at least 30days postinoculation. Median survival times were determined byinspection. The results of such a test conducted using BL5800: andBL580B appear in Table V below.

TABLE V Parasite Median survival in Diet Dose Suppression time in days X6 days mgjkgjday Day 8 (treated/controls) Compound BLMMJQ (H lit) 83"I51) 31.5301; (H 209 50 12/9 BLSSOa 0.37 673 99 l2i9 BLSSOB 0.2 350 65*15/9 Indicates possible suppression antimalarial activity Indicatessuppressive antimalarial activity.

The usefulness of BL 80a and BL580/3 is further demonstrated by their inviva anticoccidial activity in chickens according to the following test.

The invention will be described in greater detail in conjunction withthe following specific examples.

The poultry diet employed in the test is follows: EXAMPLE 1 Vitaminaminoacid premix 0.5% m l Preparation Trace mmerals 0.1% Sodium chloride 0.3%Dicalcium phosphate 12% A typical medium used to grow the primarymoculum Ground limesmne 05% was prepared according to the followingformula: Stabilized fat 4.0% Dehydrated alfalfa, 17% protein 2.0% 20Corn gluten meal, 4l% protein 5.0% Menhaden fish meal, 60% protein 5.0%Glucose 20 I Soy flour 10 gm. Soybean Oll meal, 44% protein 300/ Comsteep liquor 5 gm J... 'QFPK 'EWFE 'F m. 9 Calcium carbonate 3 gm. Watto 1000 ml The vitamin-ammo acid premlx 1n the above feed er CPmPOSmO" ff from, the lfonowmg a The washed or scraped spores from an agar slantof tron. The express ons 0 quantity re ate to units per klstreptomyceshygroscopic NRRL 5647 were used to lQgram of the fimshed composmon'inoculate two 500 ml. flasks containing 100 ml. each of the abovesterile medium. The flasks were placed on a Butylated hydroxy toluene125 mg. rotary shaker and agitated vigorously for 48 hours at 'g f fig288 28C. The resulting flask inoculum was transferred to vitamin D:l1100 j j a 5 gallon glass fermentor containing 12 liters of theRiboflavin same sterile medium. The glass fermentor was aerated VitaminE 2.2 l.U. Niacin 275 mg with sterile air while growth was carried outfor about Panthothenic acid 8.8 mg. 48 hours, after which-the contentswere used to seed a Choline chlorde 500 mg. 300 liter tank fermentorFolic acid 1.43 mg. V I Mcnadione sodium bisulfate 1.1 mg. Vitamin B I]meg. Ground ye l low corn, fine to 5 gm. EXAMPLE 2 Y r t r I r rFermentation A.mlXed.m9culu-m of sooqspomlateqropcystggigt Afermentation medium was prepared according to merza acervulma and asufficient number of oocysts of the following formula. Elmerza tenellato produce 85 percent to 100 percent mortality in untreated controls wasgiven to seven-dayold chicks, by direct inoculation into the crops ofall c- Cornstarch 40 gm. hicks. The chicks were given free access tofeed and g orn steep l|quor 5 gm. water during the entire test period.Two days before incai um carbonate 3 gm. oculation, medicated feed withseveral levels of drug water 1000 was presented to the various groups ofchicks. Seven 0 days after inoculation the tests were terminated and theT fermentatlo" medlum was stenllzed at 120 0 birds were weighed,necropsied and their intestinal .W stsamatZO b l 9O m h P tractsexamined for lesions. The results appear in Table of medmm after wasThree v below These results Show that 00 percent Survival dred liters ofthis sterile medium in a 400 liter tank ferf i f d hi k i b i d h 100ppm f mentor was inoculated with 12 liters of inoculum pre- BL5800: and[25 ppm. or 250 ppm. of BL58OB is adpared as described in Example 1. Thefermentation was ministered to infected chicks in their diet. Theselevels carried out at 28C. using Hodag LG-8 oil as a defoamalso show asignificant suppression of lesions due to E. ing agent. Aeration wassupplied at the rate of 0.5 liter tenella and E. acervulina. Y ofsterile air per liter of mash per minute. The mash was TABLE VI Birdswith Concentration No. of Birds Reduced Lesions in diet ppm. StartedSurvival E. lenella E. acervulina BL580fl Uninl'ected 0 40 100 Infected0 40 32 0 0 250 5 100 5 100 40 I00 BLSSOa Uninfected 0 20 100 Infected 020 25 0 0 100 5 100 so 100 agitated byan At the end of about 116 hoursof fermentation time the mash was harvested.

EXAMPLE 3 Isolation and Purification A 300 liter portion of fermentedmash, prepared as described in Example 2, having a pH of 7.1 wasfiltered with the aid of diatomaceous earth. The filter cake was washedwith 30 liters of tap water. The combined filtrate and washingsconsisting of about 260 liters was stirred with 130 liters of ethylacetate for 20 minutes. The mixture was allowed to settle, the phaseswere sep arated and the 125 liters ethyl acetate phase was concentratedin a still at reduced pressure to 3.2 liters which was then allowed toevaporate to an oily residue.

A 300 gm. portion of silica gel (Davison Chemical Company, Grade No. 62)was slurried in methylene chloride and the suspension was poured into a2 /2 inches diameter glass column. The excess solvent was allowed todrain away. The crude oily residue from the ethyl acetate extraction wasdissolved in a minimal quantity of methylene chloride and allowed toseep into the column of gel. The column was treated with 2,475 ml. ofmethylene chloride followed by 1,200 ml. of a methylene chloride-ethylacetate (1:1 v/v) mixture. A total of 49 fractions with a volume of 75ml. each were collected. A separate paper disc (one-fourth inchdiameter) was dipped into each fraction, air dried and bioautographed ona large agar plate seeded with @Izsntesecswzxesszw NY Erasttqns] to 37,.9911 .1 prising the active band, were combinedand concen' trated to aresidue weighing 7.08 gm.

A two-phase system was prepared by mixing hexanezethylacetate:methanol:water (28:2:16:l). A 160 gm. portion of acid-washeddiatomaceous earth was wetted with 120 ml. of the aqueous phase and thiswas packed in increments into a glass column 1 A inch in diameter. Thecharge consisted of a mixture of 16 gm. diatomaceous earth, 12 ml. oflower phase and the 7.08 gm. of partially purified product from thesilica gel chromatography. The column was developed with upper phase.Fractions of 20 ml. each were collected using an automatic collector.The active components were detected by chromatography on thin layerchromatography plates [Silica gel P 254, Merck AG- Darmstadt (Germany)]using chloroform-ethyl acetate (1:1 v/v) as a developing solvent. Thezones were detected by first spraying with sulfuric acid followed byheating on a hot plate. The active components appeared as black 1195 t RalwssfO-Mtfikfitlqd and 0.27 (BLSSOB). Fractions 4 to- 14 incusive,containing BL 80a, were combined and concentrated to a residue weighing799 mg. Similarly, fractions 24 to 65, comprising BLSSOB, wereconcentrated to a residue EXAMPLE 4 Preparation of Highly PurifiedBL5800:

Amorphous, essentially pure 131.5800; was obtained from materialprepared as described in. Example 3 (fractions 4-14) from a partitioncolumn by solvent countercurrent distribution using a system composed ofhexanezethyl acetate:methanol:water (56:12:32:3 by

. sig r gL S mg.

volume). The countercurrent distribution was conducted in a ZOO-tubeapparatus using 10 ml. of lower phase and 10 ml. of upper phase pertube. The charge consisting of 999 mg. was dissolved in 40 ml. of lowerphase and transferred into tubes l to 4. The apparatus was run for 190transfers. BL580a was found in tubes 60 to 100 by thin layerchromatography. The contents of tubes 60-100 were combined andconcentrated to an aqueous suspension containing a white precipitate.The precipitate was collected on a funnel, washed with water and driedin vacuo over P 0 at room tempera ture yielding 348 mg. of BLSSOoz.Elemental analysis: C, 62.05; H, 8.86;.N, 0.00; O(direct), 24.75; Ash,5.90. Molecular Weight (isothermal distillation in CHCI 711. SpecificRotation: [a],, l5.55 i 0.18 (C 1.08 in methanol). A standard infraredabsorption spectrum of BL580a prepared in a KBr pellet is shown in FIG.1 of the accompanying drawings.

u EXAMPLE 5 Preparation of Highly Purified BL58OB Amorphous, essentiallypure 81.5805 was obtained from material prepared as described in Example3 (fractions 24-65) from a partition column by solvent countercurrentdistribution using hexanezethyl acetate:methanol:water (280::l60:7 byvolume). The countercurrent distribution was conducted as described inExample 4 using 240 transfers and a charge of 1.3 gm. Fractions 55 to90, containing BLSSOB gave 862 mg. of a white amorphous solid uponconcentration, ly'ophilization in t-butanol, and drying in vacuo over P0 at room temperature. Elemental analysis: C, 60.74; H, 8.73; N, 0.00; 0(direct), 22.71; Ash, 5.60. .Molecular Weight (isothermal distillationin CHCI 641. Specific rotation: [04 0: 02 (C 0.902 in methanol) and[01], 1.06 i 0.2 (C= 0.942 in chlorform). A standard infrared absorptionspectrum of BL580B prepared in a KBr pellet is shown in FIG. 2 of theaccompanying drawings. We claim:

ll. Antibiotic BL580a, a compound which a. is effective in inhibitingthe growth of bacteria; and

in its essentially pure crystalline form b. has an optical rotation[a],, l5.55 i 018 (C=l.08 in methanol); 7 c. has the following elementalanalysis (percent):

C,62.05; 11,8.86; 0,2475; ash, 5.90; d. has a molecular weight of 71 las measured by isothermal distillation in chloroform; and c. has acharacteristic infrared absorption spectrum as shown in FIG. 1' of thedrawings. 2. A compound as defined in claim 1, antibiotic 131.5 a, inits essentially pure form.

3. Antibiotic 181.5803, a compound which a. is effective in inhibitingthe growth of bacteria; and

in its essentially pure crystalline form; b. has an optical rotation[01],, 1.06 i 0.2 wfl (C=0.942 in chloroform); c. has the followingelemental analysis (percent):

C,60.74; H, 8.73; 0,2271; ash, 5.60; -.5 ass. 2l.. .q .lar sbtp fislasms su sd by thermal distillation in chloroform; and e. has acharacteristic infrared absorption spectrum as shown in FIG. 2 of thedrawings. 4. A compound as defined in claim 3, antibiotic BLSSOBJn itsessentially pure form.

. :k :I: it: :1: :11

2. A compound as defined in claim 1, antibiotic BL580 Alpha , in its essentially pure form.
 3. Antibiotic BL580 Beta , a compound which a. is effective in inhibiting the growth of bacteria; and in its essentially pure crystalline form; b. has an optical rotation ( Alpha )D25 1.06* + or - 0.2* (C 0.942 in chloroform); c. has the following elemental analysis (percent): C,60.74; H, 8.73; O,22.71; ash, 5.60; d. has a molecular weight of 641 as measured by isothermal distillation in chloroform; and e. has a characteristic infrared absorption spectrum as shown in FIG. 2 of the drawings.
 4. A compound as defined in claim 3, antibiotic BL580 Beta , in its essentially pure form. 